Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes
ABSTRACT As the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-06-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02557-21 |
| _version_ | 1811238495024316416 |
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| author | Changqing Liu Jin Lei Min Zhang Fan Wu Mingdong Ren Jinsheng Yang Qinglong Wu Xiaoli Shi |
| author_facet | Changqing Liu Jin Lei Min Zhang Fan Wu Mingdong Ren Jinsheng Yang Qinglong Wu Xiaoli Shi |
| author_sort | Changqing Liu |
| collection | DOAJ |
| description | ABSTRACT As the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and counting by flow cytometry (FCM) in the laboratory. Here, we explored the effects of different preservation methods on the PPE community by combining FCM sorting and high-throughput sequencing. Our results showed that dimethyl sulfoxide (DMSO) cryoprotection could destroy the fluorescence and cell structure of the PPEs, making the subsequent FCM analysis and sorting difficult. Aldehyde fixation maintained the PPE fluorescence, and the fixed samples were of sufficient quality for abundance analysis and sorting by FCM. However, the sequencing results showed that, after preservation by aldehydes, the proportion of PPEs dramatically decreased to approximately 10%, in comparison to 90% in the fresh samples, and the sequences of Ascomycota significantly increased. In contrast, preservation with Pluronic F68 (F68) not only could maintain the PPE abundance close to the initial value but also could keep the PPE community similar to that in the fresh samples over a storage time of 6 months. Thus, F68 cryopreservation is a suitable preservation method for PPE communities from freshwater lakes. IMPORTANCE PPEs contribute significantly to primary productivity in freshwater ecosystems. The combination of FCM sorting and high-throughput sequencing has been shown to be a powerful approach and can largely improve our view of the PPE diversity. However, the water samples could not be counted and sorted immediately after sampling from many lakes due to the inaccessibility of FCM in the field. Thus, the comparison of different preservation methods that allow subsequent analysis of the community structure by high-throughput sequencing is an urgent need. Our results indicated that F68 cryopreservation could maintain the PPE abundance close to the initial value and keep the community similar to that in the fresh samples over a storage time of 6 months. |
| first_indexed | 2024-04-12T12:42:31Z |
| format | Article |
| id | doaj.art-28525b03149f40e09aeae8b399cb0ceb |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-12T12:42:31Z |
| publishDate | 2022-06-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-28525b03149f40e09aeae8b399cb0ceb2022-12-22T03:32:44ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-06-0110310.1128/spectrum.02557-21Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in LakesChangqing Liu0Jin Lei1Min Zhang2Fan Wu3Mingdong Ren4Jinsheng Yang5Qinglong Wu6Xiaoli Shi7State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaState Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinaABSTRACT As the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and counting by flow cytometry (FCM) in the laboratory. Here, we explored the effects of different preservation methods on the PPE community by combining FCM sorting and high-throughput sequencing. Our results showed that dimethyl sulfoxide (DMSO) cryoprotection could destroy the fluorescence and cell structure of the PPEs, making the subsequent FCM analysis and sorting difficult. Aldehyde fixation maintained the PPE fluorescence, and the fixed samples were of sufficient quality for abundance analysis and sorting by FCM. However, the sequencing results showed that, after preservation by aldehydes, the proportion of PPEs dramatically decreased to approximately 10%, in comparison to 90% in the fresh samples, and the sequences of Ascomycota significantly increased. In contrast, preservation with Pluronic F68 (F68) not only could maintain the PPE abundance close to the initial value but also could keep the PPE community similar to that in the fresh samples over a storage time of 6 months. Thus, F68 cryopreservation is a suitable preservation method for PPE communities from freshwater lakes. IMPORTANCE PPEs contribute significantly to primary productivity in freshwater ecosystems. The combination of FCM sorting and high-throughput sequencing has been shown to be a powerful approach and can largely improve our view of the PPE diversity. However, the water samples could not be counted and sorted immediately after sampling from many lakes due to the inaccessibility of FCM in the field. Thus, the comparison of different preservation methods that allow subsequent analysis of the community structure by high-throughput sequencing is an urgent need. Our results indicated that F68 cryopreservation could maintain the PPE abundance close to the initial value and keep the community similar to that in the fresh samples over a storage time of 6 months.https://journals.asm.org/doi/10.1128/spectrum.02557-21photosynthetic picoeukaryotesflow cytometrypreservation methodsPluronic F68 |
| spellingShingle | Changqing Liu Jin Lei Min Zhang Fan Wu Mingdong Ren Jinsheng Yang Qinglong Wu Xiaoli Shi Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes Microbiology Spectrum photosynthetic picoeukaryotes flow cytometry preservation methods Pluronic F68 |
| title | Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes |
| title_full | Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes |
| title_fullStr | Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes |
| title_full_unstemmed | Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes |
| title_short | Optimization of Preservation Methods Provides Insights into Photosynthetic Picoeukaryotes in Lakes |
| title_sort | optimization of preservation methods provides insights into photosynthetic picoeukaryotes in lakes |
| topic | photosynthetic picoeukaryotes flow cytometry preservation methods Pluronic F68 |
| url | https://journals.asm.org/doi/10.1128/spectrum.02557-21 |
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